The structural analysis of a protein by the NMR spectrometry should always be carried out while taking into consideration such problems as any possible overlapping between NMR signals and the reduction of signal intensities due to the relaxation phenomenon. In this respect, it would be essential to the solution of this problem to develop an advanced NMR measurement and analysis technique. However, proteins each having a molecular weight on the order of about 20,000 can presently be analyzed without being accompanied by any significant error because of the application of the multi-nuclear and multi-dimensional NMR spectroscopic technique developed in the early 1990s to the protein and the development of a technique for the mass-production of stable isotope-labeled proteins, which has been advanced along with the progress of the NMR spectroscopic technique.
However, all of these methods are ones for obtaining information on the three-dimensional structure of a high molecular weight protein at the sacrifice of the precision of the determination of the higher-order structure thereof. Therefore, these techniques are limited in the subject to be analyzed and the effectiveness thereof In this respect, Patent Document 1 specified below discloses an invention which can solve these conventional problems, which permits the deuterium-exchange of a protein without adversely affecting the sensitivity of the remaining hydrogen nuclei to the NMR spectroscopic measurement and which simultaneously permits the rapid and highly reliable analysis of the NMR spectra observed for a protein having a molecular weight higher than the conventional limit and the determination of the higher-order structure thereof with a high accuracy. However, this invention never specifies the isotope-labeling pattern on the aromatic ring portion present in an aromatic amino acid.
On the other hand, aromatic amino acids such as Phe, Tyr and Trp play important roles along with the amino acids each carrying a long chain alkyl group such as Leu, Val and Ile in the formation of the three-dimensional structure of the hydrophobic core portion contained in a globular protein. In addition, these aromatic amino acids likewise play important roles in the manifestation of protein functions typical of the substrate-recognizing function, while making the most use of functional groups such as the hydroxyl group of Tyr and the nitrogen derived from the indole ring of lip, or the π-electrons common to the aromatic rings, in addition to the roles in the formation of the three-dimensional structure. In this respect, however, if using a sample uniformly labeled with stable isotope (13C, 15N, 2H) disclosed in Patent Document 1 or a sample (non-labeled) having a natural abundance ratio of isotopes, the proton NMR signals, ascribed to the ring portions of the aromatic amino acids, in particular, Phe and Th), show chemical shifts quite close to one another and the chemical shifts of the carbon atoms (13C) to which they are bonded likewise come close to one another. Accordingly, quite complicated signals are obtained for such a uniformly labeled derivative, this accordingly results in the deterioration of the sensitivity thereof to the NMR spectroscopic measurement and this makes, quite difficult, the individual observation of signals and the assignment thereof to each corresponding sequence.
Under such circumstances, there have been proposed a variety of methods for overcoming these difficulties, for collecting the information of nuclear Overhauser effects (NOE) concerning aromatic ring protons serving as the distance-limiting information quite important for the determination of the three-dimensional structure and for accurately measuring information on the local structure of aromatic rings. However, all of the conventional methods are ones developed while aiming at the sample uniformly labeled with stable isotope (13C, 15N, 2H), whose preparation is quite easy and therefore, there has not yet been developed any method quite excellent from the viewpoint of the practicability.    Patent Document 1: International Publication WO 03/053910A1